CN105158486A - Enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein) - Google Patents
Enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein) Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention belongs to the field of medical detection, particularly relates to an enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein). The linked immunosorbent assay kit comprises components as follows: (1), an elisa plate coated with human ox-LDL antibodies; (2), standard products in human ox-LDL series; (3), an enzyme-labeled antibody solution; (4), a diluent; (5), a washing liquid; (6), a substrate solution; (7), a developing solution; (8), a stop solution.
Description
Technical field
The invention belongs to medical detection field, being specifically related to a kind of for the enzyme linked immunological kit for detecting people's OxLDL ELISA.
Background technology
Atherosclerotic (atherosclerosisAS) has referred to that arterial wall thickens, hardening and elasticity reduction, and forms with endarterium the pathology that atheromatous plaque is feature.At present its pathogenic factor is determined not yet completely, may be relevant with factors such as age, sex, dyslipidemia, hypertension, smoking, diabetes, obesity, infection.High cholesterol concentration, especially low-density lipoprotein (LDL) are the main risk factors of of AS.Recent study finds, after oxidative modification, it causes AS effect and greatly strengthens low-density lipoprotein.Think at present OxLDL ELISA (Oxidizedlowdensitylipoprotein, ox-LDL) mainly from endothelial cell damage, promote foam wanshing, promote the propagation of vascular smooth muscle cell, cause the aspect such as platelet adhesion reaction and gathering to participate in atherosclerotic formation.The core component of the oxidative modification LDL of 1-LDL is triglyceride and cholesterol ester, and surface component is apolipoprotein, free cholesterol and phosphatide.LDL contains abundant polyunsaturated fatty acid, brings out a large amount of oxygen radical of lower generation in factors such as smoking, medicine, hypertension, diabetes, is easy to oxidizedly be modified into OxLDL ELISA (ox-LDL).Coronary heart disease, coronary atherosclerotic heart disease, main typical history, the clinical symptoms according to being patient of its diagnosis, and carry out in conjunction with instrument and biochemical analysis.Detection methods conventional at present has routine electrocardiogram, electrocardiogram stress test, echocardiogram, spiral CT and UFCT Ultra-fast Computed Tomograph, coronary artery ultrasonoscopy, intravascular ultrasound, Nuclear Cardiac Imaging and selective coronary arteriography, and laboratory examination comprises Myocardial Enzymologic inspection, troponin, c reactive protein, blood fat and lipoprotein inspection etc.In above detection methods, great majority are electrocardiosignal change (various cardiogram) and ventricular wall motion metamorphosis (echocardiogram after detecting myocardial ischemia or myocardial infarction, cardiac radionuclide measurement, spiral CT and UFCT Ultra-fast Computed Tomograph, magnetic resonance imaging) based on carry out the diagnosis of coronary heart disease, if degree of stenosis not yet causes myocardial ischemia, above detection means often can not contribute to diagnosis of coronary heart disease.
Research shows, OxLDL ELISA Ox-LDL develops relevant with the generation of coronary heart disease, it has been generally acknowledged that and by detecting the content of OxLDL ELISA Ox-LDL in sample serum to be checked, can assess the risk suffering from coronary heart disease.In prior art, disclose the detection method that some are relevant, as: number of patent application: 03109935.1, denomination of invention: with anti-phosphocholine antibody to the quantitative measurement of OxLDL ELISA and the application in diagnosing atherosclerotic thereof.The quantitative detecting method of this disclosure of the invention comprises the following steps: that (a) is by the antibody of anti-phosphocholine and the sample contacts containing Ox-LDL; B () makes described antibody be combined with Ox-LDL; C () measures the binding capacity of described antibody and Ox-LDL; D () foundation is the typical curve that standard items record with phosphocholine, quantitative Ox-LDL content.Because antigen-antibody reaction is very complicated, relevant with several factors, although the epiope of phosphocholine is identical with the epiope that low-density lipoprotein presents through oxidative modification rear surface, but the degrees of specificity of the antigen-antibody reaction of anti-phosphocholine antibody and OxLDL ELISA is difficult to predict, the accuracy detecting OxLDL ELISA with anti-phosphocholine antibody is not high enough, is difficult to be applied to clinical detection.
Patent CN201210160797 discloses a kind of ELISA detection kit of people's OxLDL ELISA, this kit has good sensitivity and accuracy, but this stabilization of kit is not good in practical application, and monoclonal antibody output is lower, affects its large-scale application.
Summary of the invention
A first aspect of the present invention is to provide a kind of enzyme-linked immunologic detecting kit of people's OxLDL ELISA, and it consists of the following composition: (1) is coated with the ELISA Plate of anti-human OxLDL ELISA antibody; (2) people's OxLDL ELISA serial standards; (3) enzyme mark antibody solution; (4) dilution; (5) cleansing solution; (6) substrate solution; (7) nitrite ion; (8) stop buffer.
People's OxLDL ELISA content of described people's OxLDL ELISA serial standards is: 0,1,2,4,8 and the 15mmol/LPBS damping fluid of 16U/L, and containing 1g casein and 5g sucrose in often liter of standard solution.
Described enzyme mark antibody solution is the 15mmol/LPBS damping fluid of the PEG200 of anti-apoB antibody (goat-anti human apolipoprotein b antibody) and the 0.2mg/L marked containing the HRP of 0.5mg/L, and pH value is 8.5.
Described dilution is PBS (pH7.4) damping fluid of 15mmol/L;
Described cleansing solution is the 0.05% polysorbas20 solution that the PBS (pH7.4) of 15mmol/L prepares.
Described substrate solution is 3% superoxol that phosphoric acid-citrate buffer solution (pH7.4) is prepared, and the Sodium Acid Pyrophosphate of 0.1mg/L in solution;
Described nitrite ion is the methanol solution of tetramethyl benzidine (TMB), and concentration is 0.1mg/ml;
Described stop buffer is 3mol/L sulfuric acid.
A second aspect of the present invention is to provide the preparation method of described kit, and concrete steps are as follows:
(1) preparation of anti-human OxLDL ELISA antibody: will hybridoma (preserving number the is CCTCCNO:C200304) in vitro culture of anti-OxLDL monoclonal antibody be expressed; Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1; 3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times.And during cell chulture, add 10IU/10mlLIF(leukocyte inhibitory factor in the medium), 2IU/10mlHCG, 2IU/10ml bovine insulin.Culture supernatant, through filtering clarification, purifying, is collected antibody and get final product;
(2) preparation of the ELISA Plate of anti-human OxLDL ELISA antibody is coated with:
A, antibody dilution: be that anti-human OxLDL ELISA monoclonal antibody is diluted to 2.5 20 μ g/ml by the Tris-HCl buffer solution of the 50mM of 8.0 with pH, obtain coating buffer;
B, bag quilt: get microwell plate, wash 3 times with cleansing solution, and add the above-mentioned coating buffer containing anti-human OxLDL ELISA monoclonal antibody, μ L/ hole, every hole 100, hatches 12 hours for 4 DEG C;
C, to close: incline coating buffer, is placed on thieving paper and pats several times, removes raffinate, the concentration added containing 1% casein and 1% sucrose is the Tris-HCl confining liquid of 50mmol/L, and its pH is 8.0,300 μ l/ holes, room temperature, 1 hour;
D, vacuum drying, sealing, must be coated with the ELISA Plate of anti-human OxLDL ELISA antibody.
(3) enzyme mark antibody solution preparation:
A, to be dissolved in 10mgHRP in 1ml distilled water and to add freshly prepared 0.06mol/LNaIO
41ml, mixes and places 30 minutes in 4 DEG C;
B, add 0.16mol/L glycol water 1ml, room temperature places 30 minutes;
C, adds the aqueous solution 2ml containing the anti-apoB antibody of 5mg, then at 4 DEG C, to 0.05mol/L carbonic acid buffer (pH9.6) dialysed overnight;
D, solution in sucking-off bag filter, adds 0.5mlNaBH4, places 2 hours at 4 DEG C;
E, drips equal-volume saturated (MM) 2SO4 solution, places 30 minutes at 4 DEG C;
Centrifugal 10 minutes of f, above-mentioned solution 2500rpm, remove supernatant, precipitate and dissolve with the PBS of a little 0.02mol/LpH7.4, and to this damping fluid dialysed overnight at 4 DEG C;
G, solution in sucking-off bag filter, centrifugal removing insolubles, Superose6 chromatographic column crossed by supernatant, with the PBS wash-out of 0.02mol/LpH7.4, collects first peak, is the enzymic-labelled antibody of purifying;
H, by the enzymic-labelled antibody aseptic filtration of collecting, then with containing the 15mmol/LPBS buffer solution of PEG200 of 0.2mg/L to antibody final concentration 0.5mg/L.
(4) preparation of people's OxLDL ELISA standard items:
The preparation of a, LDL: be separated human plasma lipoprotein by disposable density－gradient centrifuga－tion method, collection density is 1.03 to LDL component l.05g/ml,
The preparation of b, Ox-LDL: external acellular Ni metal
2+ox-LDL, for OxLDL, then dilutes according to respective concentration water by ionic oxide formation legal system, and in often liter of standard solution, add 1g/L casein and 5g/L sucrose.
(5) dilution, cleansing solution, substrate solution, nitrite ion and stop buffer, prepares according to the solution preparation method of this area routine.
Embodiment
Further will illustrate the present invention below.It is pointed out that following explanation is only illustrating the technical scheme that application claims is protected, any restriction not to these technical schemes.The content that protection scope of the present invention is recorded with appended claims is as the criterion.
embodiment 1
(1) preparation of anti-human OxLDL ELISA antibody: will hybridoma (preserving number the is CCTCCNO:C200304) in vitro culture of anti-OxLDL monoclonal antibody be expressed; Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1; 3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times.And during cell chulture, add 10IU/10mlLIF(leukocyte inhibitory factor in the medium), 2IU/10mlHCG, 2IU/10ml bovine insulin.Culture supernatant, through filtering clarification, purifying, is collected antibody and get final product;
(2) preparation of the ELISA Plate of anti-human OxLDL ELISA antibody is coated with:
A, antibody dilution: be that anti-human OxLDL ELISA monoclonal antibody is diluted to 2.5 20 μ g/ml by the Tris-HCl buffer solution of the 50mM of 8.0 with pH, obtain coating buffer;
B, bag quilt: get microwell plate, wash 3 times with cleansing solution, and add the above-mentioned coating buffer containing anti-human OxLDL ELISA monoclonal antibody, μ L/ hole, every hole 100, hatches 12 hours for 4 DEG C;
C, to close: incline coating buffer, is placed on thieving paper and pats several times, removes raffinate, the concentration added containing 1% casein and 1% sucrose is the Tris-HCl confining liquid of 50mmol/L, and its pH is 8.0,300 μ l/ holes, room temperature, 1 hour;
D, vacuum drying, sealing, must be coated with the ELISA Plate of anti-human OxLDL ELISA antibody.
(3) enzyme mark antibody solution preparation:
A, to be dissolved in 10mgHRP in 1ml distilled water and to add freshly prepared 0.06mol/LNaIO
41ml, mixes and places 30 minutes in 4 DEG C;
B, add 0.16mol/L glycol water 1ml, room temperature places 30 minutes;
C, adds the aqueous solution 2ml containing the anti-apoB antibody of 5mg, then at 4 DEG C, to 0.05mol/L carbonic acid buffer (pH9.6) dialysed overnight;
D, solution in sucking-off bag filter, adds 0.5mlNaBH4, places 2 hours at 4 DEG C;
E, drips equal-volume saturated (MM) 2SO4 solution, places 30 minutes at 4 DEG C;
Centrifugal 10 minutes of f, above-mentioned solution 2500rpm, remove supernatant, precipitate and dissolve with the PBS of a little 0.02mol/LpH7.4, and to this damping fluid dialysed overnight at 4 DEG C;
G, solution in sucking-off bag filter, centrifugal removing insolubles, Superose6 chromatographic column crossed by supernatant, with the PBS wash-out of 0.02mol/LpH7.4, collects first peak, is the enzymic-labelled antibody of purifying;
H, by the enzymic-labelled antibody aseptic filtration of collecting, then with containing the 15mmol/LPBS buffer solution of PEG200 of 0.2mg/L to antibody final concentration 0.5mg/L.
(4) preparation of people's OxLDL ELISA standard items:
The preparation of a, LDL: be separated human plasma lipoprotein by disposable density－gradient centrifuga－tion method, collection density is 1.03 to LDL component l.05g/ml,
The preparation of b, Ox-LDL: external acellular Ni metal
2+ox-LDL, for OxLDL, then dilutes according to PBS (pH7.4) damping fluid of respective concentration 15mmol/L, and in often liter of standard solution, adds 1g/L casein and 5g/L sucrose by ionic oxide formation legal system.
(5) dilution, cleansing solution, substrate solution, nitrite ion and stop buffer, prepares according to the solution preparation method of this area routine.
People's OxLDL ELISA content of described people's OxLDL ELISA serial standards is: 0,1,2,4,8 and the 15mmol/LPBS damping fluid of 16U/L, and containing 1g casein and 5g sucrose in often liter of standard items.
Described enzyme mark antibody solution is the 15mmol/LPBS damping fluid of the PEG200 of anti-apoB antibody (goat-anti human apolipoprotein b antibody) and the 0.2mg/L marked containing the HRP of 0.5mg/L, and pH value is 8.5.
Described dilution is PBS (pH7.4) damping fluid of 15mmol/L;
Described cleansing solution is the 0.05% polysorbas20 solution that the PBS (pH7.4) of 15mmol/L prepares.
Described substrate solution is 3% superoxol that phosphoric acid-citrate buffer solution (pH7.4) is prepared, and the Sodium Acid Pyrophosphate of 0.1mg/L in solution;
Described nitrite ion is the methanol solution of tetramethyl benzidine (TMB), and concentration is 0.1mg/ml;
Described stop buffer is 3mol/L sulfuric acid.
embodiment 2 kit sensitivity determination
Prepare the PBS damping fluid of Ox-LDL standard items variable concentrations respectively, concentration is respectively 0.1U/L, 0.5U/L, 1U/L, 5U/L, 10U/L, 20U/L, the kit adopting embodiment 1 to prepare detects, to contrast damping fluid as blank, and use comparative example kit to detect equally, comparative example is ELISA kit prepared by patent CN201210160797 embodiment 1, and concrete detection method is as follows simultaneously:
A) antigen-antibody reaction: add 50 μ l standard items standard solution and dilutions in the micropore of coated elisa plate respectively, 37 DEG C of water bath heat preservations 50 minutes.Cleaning buffer solution is washed plate and is operated 5 times.
B) the anti-apoB antibody-solutions that HRP marks is added each hole, every hole 100 μ l, 37 DEG C of water bath heat preservations 50 minutes.Repeat to wash plate and operate 5 times.
C) chromogenic reaction: every hole adds substrate solution, each 50 μ l of nitrite ion successively, 37 DEG C of water bath heat preservations 20 minutes, every hole adds 50 μ l reaction terminating liquids again and terminates reaction.
D) colorimetric: measure OD value and record at 450nm by microplate reader.
E) production standard curve: take standard concentration as horizontal ordinate, the OD value that standard items measure is ordinate, makes typical curve; Calculate typical curve regression coefficient R
2, work as R
2during > 0.99, this measures effectively;
Calculate the ratio of Ox-LDL standard items and blank, when ratio is greater than 2, illustrate that kit can measure the Ox-LDL standard items of this concentration, least concentration is the sensitivity of kit, and parallel experiment is averaged for five times, and concrete outcome is as follows:
Result shows that its sensitivity of kit prepared by the embodiment of the present invention 1 can reach 0.1U/L, and the sensitivity of comparative example is 0.5U/L.And obtain absorbance data according to variable concentrations standard items to return, obtaining regression equation is y=0.237x-0.0032; R
2=0.99.
embodiment 3
Anti-human OxLDL ELISA antibody is prepared according to the method for patent CN201210160797 embodiment 1, antibody content before employing HPLC method mensuration purifying in supernatant, same mensuration is carried out to the embodiment of the present invention 1 simultaneously, result shows: in the supernatant that the embodiment of the present invention 1 is gathered in the crops, antibody content is 9.7mg/L, and in the supernatant of patent CN201210160797 embodiment 1, antibody content is 3.8mg/L.
embodiment 4 stabilization of kit is investigated
After kit embodiment 1 prepared places 6 months and 12 months respectively at 20 DEG C, measure the sensitivity of kit according to the method for embodiment 2, and regretional analysis is carried out to data, calculate R
2value.
In the present embodiment, comparative example is set as follows:
Comparative example 1: the preparation method of kit is with embodiment 1, and difference is only not add PEG200 in the anti-apoB antibody-solutions that HRP marks.
Comparative example 2: the preparation method of kit is with embodiment 1, and difference is only that in the anti-apoB antibody-solutions that HRP marks, PEG200 replaces with hyclone (FBS), the same PEG200 of concentration.
Comparative example 3: the preparation method of kit is with embodiment 1, and difference is only in standard items PBS solution, containing the sucrose of BSA and 50g/L of 20g/L.
Comparative example 4: the preparation method of kit is with embodiment 1, and difference is only in standard items PBS solution, containing the casein of 0.5g/L and the sucrose of 2g/L.
Comparative example 5: the preparation method of kit is with embodiment 1, and difference is only in standard items PBS solution, containing the casein of 3g/L and the sucrose of 10g/L.
Concrete outcome is as follows:
In addition, preserve after 36 months at 4 DEG C, kit sensitivity and linearly good, with the kit just prepared without significant difference.
embodiment 5
The quality testing precision of enzyme-linked immune quantitative detection reagent box prepared by the application embodiment of the present invention 1: randomly draw 50 box different batches kits, carry out replication with a atherosclerotic's serum by specification operation steps.Calculate each measurement result, obtain average, SD and coefficient of variation CV.Precision test result display batch between CV be less than 2%.
Content of the present invention merely illustrates some claimed specific embodiments; one of them or more described technical characteristic can be combined with arbitrary one or more technical scheme in technical scheme; these technical schemes obtained through combination also in the application's protection domain, just as these technical schemes obtained through combination in the disclosure of invention concrete record.
Claims (7)
1. an enzyme-linked immunologic detecting kit for people's OxLDL ELISA, it consists of the following composition: (1) is coated with the ELISA Plate of anti-human OxLDL ELISA antibody; (2) people's OxLDL ELISA serial standards; (3) enzyme mark antibody solution; (4) dilution; (5) cleansing solution; (6) substrate solution; (7) nitrite ion; (8) stop buffer.
2. enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that, people's OxLDL ELISA content of described people's OxLDL ELISA serial standards is: 0,1,2,4,8 and the 15mmol/LPBS damping fluid of 16U/L, and containing 1g casein and 5g sucrose in often liter of standard solution.
3. enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that, described enzyme mark antibody solution is the 15mmol/LPBS damping fluid of the PEG200 of anti-apoB antibody (goat-anti human apolipoprotein b antibody) and the 0.2mg/L marked containing the HRP of 0.5mg/L, and pH value is 8.5.
4. enzyme-linked immunologic detecting kit according to claim 1, is characterized in that, described dilution is PBS (pH7.4) damping fluid of 15mmol/L; Described cleansing solution is the 0.05% polysorbas20 solution that the PBS (pH7.4) of 15mmol/L prepares.
5. enzyme-linked immunologic detecting kit according to claim 1, is characterized in that, described substrate solution is 3% superoxol that phosphoric acid-citrate buffer solution (pH7.4) is prepared, and the Sodium Acid Pyrophosphate of 0.1mg/L in solution.
6. enzyme-linked immunologic detecting kit according to claim 1, is characterized in that, described nitrite ion is the methanol solution of tetramethyl benzidine (TMB), and concentration is 0.1mg/ml; Described stop buffer is 3mol/L sulfuric acid.
7. a preparation method for enzyme-linked immunologic detecting kit according to claim 1, concrete steps are as follows:
(1) preparation of anti-human OxLDL ELISA antibody: will hybridoma (preserving number the is CCTCCNO:C200304) in vitro culture of anti-OxLDL monoclonal antibody be expressed; Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1; 3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times; And during cell chulture, add 10IU/10mlLIF(leukocyte inhibitory factor in the medium), 2IU/10mlHCG, 2IU/10ml bovine insulin; Culture supernatant, through filtering clarification, purifying, is collected antibody and get final product;
(2) preparation of the ELISA Plate of anti-human OxLDL ELISA antibody is coated with:
A, antibody dilution: be that anti-human OxLDL ELISA monoclonal antibody is diluted to 2.5 20 μ g/ml by the Tris-HCl buffer solution of the 50mM of 8.0 with pH, obtain coating buffer;
B, bag quilt: get microwell plate, wash 3 times with cleansing solution, and add the above-mentioned coating buffer containing anti-human OxLDL ELISA monoclonal antibody, μ L/ hole, every hole 100, hatches 12 hours for 4 DEG C;
C, to close: incline coating buffer, is placed on thieving paper and pats several times, removes raffinate, the concentration added containing 1% casein and 1% sucrose is the Tris-HCl confining liquid of 50mmol/L, and its pH is 8.0,300 μ l/ holes, room temperature, 1 hour;
D, vacuum drying, sealing, must be coated with the ELISA Plate of anti-human OxLDL ELISA antibody;
(3) enzyme mark antibody solution preparation:
A, to be dissolved in 10mgHRP in 1ml distilled water and to add freshly prepared 0.06mol/LNaIO
41ml, mixes and places 30 minutes in 4 DEG C;
B, add 0.16mol/L glycol water 1ml, room temperature places 30 minutes;
C, adds the aqueous solution 2ml containing the anti-apoB antibody of 5mg, then at 4 DEG C, to 0.05mol/L carbonic acid buffer (pH9.6) dialysed overnight;
D, solution in sucking-off bag filter, adds 0.5mlNaBH4, places 2 hours at 4 DEG C;
E, drips equal-volume saturated (MM) 2SO4 solution, places 30 minutes at 4 DEG C;
Centrifugal 10 minutes of f, above-mentioned solution 2500rpm, remove supernatant, precipitate and dissolve with the PBS of a little 0.02mol/LpH7.4, and to this damping fluid dialysed overnight at 4 DEG C;
G, solution in sucking-off bag filter, centrifugal removing insolubles, Superose6 chromatographic column crossed by supernatant, with the PBS wash-out of 0.02mol/LpH7.4, collects first peak, is the enzymic-labelled antibody of purifying;
H, by the enzymic-labelled antibody aseptic filtration of collecting, then with containing the 15mmol/LPBS buffer solution of PEG200 of 0.2mg/L to antibody final concentration 0.5mg/L;
(4) preparation of people's OxLDL ELISA standard items:
The preparation of a, LDL: be separated human plasma lipoprotein by disposable density－gradient centrifuga－tion method, collection density is 1.03 to LDL component l.05g/ml,
The preparation of b, Ox-LDL: external acellular Ni metal
2+ox-LDL, for OxLDL, then dilutes according to respective concentration water by ionic oxide formation legal system, and in often liter of standard solution, add 1g/L casein and 5g/L sucrose;
(5) dilution, cleansing solution, substrate solution, nitrite ion and stop buffer, prepares according to the solution preparation method of this area routine.
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| CN106248946A (en) * | 2016-08-19 | 2016-12-21 | 浙江大学 | A kind of enzyme-linked immunologic detecting kit and application thereof |
| CN106546751A (en) * | 2016-11-16 | 2017-03-29 | 广州华弘生物科技有限公司 | For detecting the enzyme linked immunological kit of OCT4 albumen |
| CN110749731A (en) * | 2019-10-18 | 2020-02-04 | 北京协和洛克生物技术有限责任公司 | Time-resolved immunochromatography kit for quantitatively detecting oxidized low-density lipoprotein and application thereof |
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| CN105974143A (en) * | 2016-04-08 | 2016-09-28 | 刘庆平 | Enzyme-linked immunosorbent assay kit for detection of human oxidized low-density lipoprotein and preparation method thereof |
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| CN111855988A (en) * | 2019-04-25 | 2020-10-30 | 常州博闻迪医药股份有限公司 | Oxidized low-density lipoprotein fluorescence detection kit and preparation method thereof |
| CN110749731A (en) * | 2019-10-18 | 2020-02-04 | 北京协和洛克生物技术有限责任公司 | Time-resolved immunochromatography kit for quantitatively detecting oxidized low-density lipoprotein and application thereof |
| CN112129946A (en) * | 2020-08-16 | 2020-12-25 | 陆修委 | Preparation method and application of sugar-free chain type inert protein sealant |
| CN117192134A (en) * | 2023-09-14 | 2023-12-08 | 宁波美康盛德生物科技有限公司 | A detection kit and detection method for oxidized low-density lipoprotein |
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